Salmonella cause 1.4 million cases of gastroenteritis and enteric fever per year in the US and lead all other foodborne bacterial pathogens as a cause of death. A prerequisite for Salmonella to cause both intestinal and systemic disease is the direct injection of effector proteins into host intestinal epithelial cells via a Type Three Secretion System (T3SS) encoded on Salmonella Pathogenicity Island 1 (SPI1). These effector proteins induce inflammatory diarrhea and bacterial invasion. Expression of the SPI1 T3SS is tightly regulated in response to environmental signals from a variety of global regulatory systems. Our long term goal is to obtain a comprehensive understanding of how the signal transduction pathways that control the SPI1 T3SS are integrated during the infection process. Extensive genetic analysis has allowed us to formulate a new model for the SPI1 regulatory circuit in which the three AraC-like regulators HilD, HilC, and RtsA act in a complex feed-forward regulatory loop to control expression of hilA, encoding the direct regulator of the SPI1 structural genes. We hypothesize that regulatory signals feed into the system primarily via post-translational control of HilD, which in turn activates hilC, rtsA, and hilA. But how these regulatory systems control HilD is unknown. The flagellar protein FliZ and the protein HilE independently control HilD at the protein level, most likely via protein-protein interaction with the N-terminal domain of HilD. As these represent proximal regulatory inputs, we focus on understanding how FliZ and HilE control HilD function. The specific aims of this proposal are to: 1. Determine how FliZ and HilE act to control HilD activity. Biochemical and genetic experiments will dissect each step in HilD activation of hilA to determine how FliZ and HilE act to control HilD function or stability. 2. Characterize the nature of the interaction between HilD and HilE or FliZ. Co-immunoprecipation and two-hybrid analysis will be used to characterize HilE-HilD and FliZ-HilD interactions. HilD point mutations that negate regulation will be used to identify regions of HilD that are specifically required for HilE- or FliZ-dependent regulation. These mutants will also allow us to test the role of FliZ- and HilE-dependent regulation of SPI1 during intestinal invasion. 3. Determine the signal transduction pathways that feed into HilD to control the SPI1 T3SS. Known regulatory systems that control SPI1 T3SS expression will be screened for those that function through HilD. Mass spectrometry and two-hybrid analysis will be used to identify additional factors that interact with HilD protein. Characterization of these factors will lead to our overall understanding of global signal transduction. The regulation of the SP1 T3SS serves as a paradigm for the integration of host environmental signals to control virulence gene expression and analysis of this system is critical to our understanding of this Class B priority pathogen.